Anthrax is a zoonotic disease transmissible between humans and animals, which is caused by Bacillus anthracis infection (Turnbull et al., WHO/EMC/ZDI, 3rd Ed. 1998). The pathogenesis of infection by B. anthracis is mainly related to the two exotoxins secreted thereby and the capsule, which are encoded on two plamids pXO1 and pXO2, respectively. The exotoxins of B. anthracis are composed of 3 different proteins; a protective antigen (PA), an edema factor (EF), and a lethal factor (LF), and these proteins are non-toxic when separated from each other. However, PA combines with either EF or LF to form an edema toxin or lethal toxin, with which protective antigens combine to facilitate the delivery of EF and LF into the cells. Further, PA is used for producing an anthrax vaccine for diagnosis or prevention because it is capable of inducing a protective antibody-response against B. anthracis infection. Accordingly, there have been numerous attempts to develop an effective method for producing PA on a large scale.
There have been reported methods for preparing such agents for preventing anthrax infection by combinant technologies using bacteria such as B. anthracis (Farchaus J W et al., Appl. Environ. Microbiol., 64, 982-991, 1998), B. subtilis (Baillie L et al., J. Appl. Microbiol., 84, 741-746, 1998) and E. coli (Laird M W et al., Protein Expr. Purif., 38, 145-152, 2004), and the usefulness of the anthrax protective antigens produced by the above methods has been described. Such recombinant anthrax protective antigens are generally purified by chromatography, and there have been many reports describing methods for purifying recombinant anthrax antigens by column chromatography. For example, Farchaus J W et al. (Appl. Environ. Microbiol., 64, 982-991, 1998) discloses a method of purifying anthrax protective antigen by ultrafiltration, ion exchange chromatography and hydrophobic chromatography; Gupta P et al. (Protein Expr. Purif., 16, 369-376, 1999), a method of purifying anthrax protective antigen by nickel affinity chromatography; and Laird M W et al. (Protein Expr. Purif., 38, 145-152, 2004), a method of purifying anthrax protective antigen by ion exchange chromatography and hydroxyapatite chromatography.
However, the above methods are uneconomical due to their complicated purification steps and the required use of expensive resins.
Therefore, there is a need for an improved method for preparing in a large scale a highly pure antigen for preventing anthrax infection, which is superior to the conventional methods in terms of maintaining the agent's immunogenicity, stability and safety.